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EC-Confetti mice treated with d-flow and hypercholesterolemia at 4 weeks post-PCL (N=6 male and 15 female) were imaged macroscopically ( A ) and LCAs/RCAs were longitudinally sectioned, stained, imaged by fluorescence microscopy ( B-R ), and quantified ( S-V ). A shows representative gross image of LCA, RCA, and aortic arch. B-R . LCAs and RCAs were immunostained with markers of endothelial inflammation (Vcam1 and Icam1, B-D ); EndMT (Acta2, Snai1, and Cnn1, E-H ); EndIT (Cd68, C1qa, C1qb, and Lyz2, I-M ); and EndFT (Spp1, Lgals3, Trem2, and BODIPY, N-R ). B, E, I, and N show merged images of confetti and FIRE markers at low magnification (10X), while the rest show 40X images. Confetti signals show eGFP (green), YFP (green), and RFP (red). All FIRE markers are shown in white except for green BODIPY ( R ). White arrows indicate confetti + ECs co-expressing the FIRE markers. S-V. Percent confetti + ECs co-expressing each FIRE marker was quantified by a combined <t>Matlab</t> and ImageJ analysis. Confetti + ECs expressing markers of inflammation (Icam1 and Vcam1, S ); EndMT (Acta2, Cnn1, and Snai1, T ); EndIT (C1qa, C1qb, Lyz2, and Cd68, U ); and EndFT (Lgals3, Trem2, and Spp1, V ). Shown are mean ± SEM, each dot represents % of confetti + ECs coexpressing FIRE markers in each longitudinal section used for quantification (N=4 to 10 longitudinal sections for RCA; N=6 to 31 longitudinal sections for LCA). P values were calculated by 2-tailed unpaired Student t test with or without Welch’s correction for normal data and 2-tailed unpaired Mann-Whitney U test for non-normal data.
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EC-Confetti mice treated with d-flow and hypercholesterolemia at 4 weeks post-PCL (N=6 male and 15 female) were imaged macroscopically ( A ) and LCAs/RCAs were longitudinally sectioned, stained, imaged by fluorescence microscopy ( B-R ), and quantified ( S-V ). A shows representative gross image of LCA, RCA, and aortic arch. B-R . LCAs and RCAs were immunostained with markers of endothelial inflammation (Vcam1 and Icam1, B-D ); EndMT (Acta2, Snai1, and Cnn1, E-H ); EndIT (Cd68, C1qa, C1qb, and Lyz2, I-M ); and EndFT (Spp1, Lgals3, Trem2, and BODIPY, N-R ). B, E, I, and N show merged images of confetti and FIRE markers at low magnification (10X), while the rest show 40X images. Confetti signals show eGFP (green), YFP (green), and RFP (red). All FIRE markers are shown in white except for green BODIPY ( R ). White arrows indicate confetti + ECs co-expressing the FIRE markers. S-V. Percent confetti + ECs co-expressing each FIRE marker was quantified by a combined <t>Matlab</t> and ImageJ analysis. Confetti + ECs expressing markers of inflammation (Icam1 and Vcam1, S ); EndMT (Acta2, Cnn1, and Snai1, T ); EndIT (C1qa, C1qb, Lyz2, and Cd68, U ); and EndFT (Lgals3, Trem2, and Spp1, V ). Shown are mean ± SEM, each dot represents % of confetti + ECs coexpressing FIRE markers in each longitudinal section used for quantification (N=4 to 10 longitudinal sections for RCA; N=6 to 31 longitudinal sections for LCA). P values were calculated by 2-tailed unpaired Student t test with or without Welch’s correction for normal data and 2-tailed unpaired Mann-Whitney U test for non-normal data.
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EC-Confetti mice treated with d-flow and hypercholesterolemia at 4 weeks post-PCL (N=6 male and 15 female) were imaged macroscopically ( A ) and LCAs/RCAs were longitudinally sectioned, stained, imaged by fluorescence microscopy ( B-R ), and quantified ( S-V ). A shows representative gross image of LCA, RCA, and aortic arch. B-R . LCAs and RCAs were immunostained with markers of endothelial inflammation (Vcam1 and Icam1, B-D ); EndMT (Acta2, Snai1, and Cnn1, E-H ); EndIT (Cd68, C1qa, C1qb, and Lyz2, I-M ); and EndFT (Spp1, Lgals3, Trem2, and BODIPY, N-R ). B, E, I, and N show merged images of confetti and FIRE markers at low magnification (10X), while the rest show 40X images. Confetti signals show eGFP (green), YFP (green), and RFP (red). All FIRE markers are shown in white except for green BODIPY ( R ). White arrows indicate confetti + ECs co-expressing the FIRE markers. S-V. Percent confetti + ECs co-expressing each FIRE marker was quantified by a combined <t>Matlab</t> and ImageJ analysis. Confetti + ECs expressing markers of inflammation (Icam1 and Vcam1, S ); EndMT (Acta2, Cnn1, and Snai1, T ); EndIT (C1qa, C1qb, Lyz2, and Cd68, U ); and EndFT (Lgals3, Trem2, and Spp1, V ). Shown are mean ± SEM, each dot represents % of confetti + ECs coexpressing FIRE markers in each longitudinal section used for quantification (N=4 to 10 longitudinal sections for RCA; N=6 to 31 longitudinal sections for LCA). P values were calculated by 2-tailed unpaired Student t test with or without Welch’s correction for normal data and 2-tailed unpaired Mann-Whitney U test for non-normal data.
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EC-Confetti mice treated with d-flow and hypercholesterolemia at 4 weeks post-PCL (N=6 male and 15 female) were imaged macroscopically ( A ) and LCAs/RCAs were longitudinally sectioned, stained, imaged by fluorescence microscopy ( B-R ), and quantified ( S-V ). A shows representative gross image of LCA, RCA, and aortic arch. B-R . LCAs and RCAs were immunostained with markers of endothelial inflammation (Vcam1 and Icam1, B-D ); EndMT (Acta2, Snai1, and Cnn1, E-H ); EndIT (Cd68, C1qa, C1qb, and Lyz2, I-M ); and EndFT (Spp1, Lgals3, Trem2, and BODIPY, N-R ). B, E, I, and N show merged images of confetti and FIRE markers at low magnification (10X), while the rest show 40X images. Confetti signals show eGFP (green), YFP (green), and RFP (red). All FIRE markers are shown in white except for green BODIPY ( R ). White arrows indicate confetti + ECs co-expressing the FIRE markers. S-V. Percent confetti + ECs co-expressing each FIRE marker was quantified by a combined <t>Matlab</t> and ImageJ analysis. Confetti + ECs expressing markers of inflammation (Icam1 and Vcam1, S ); EndMT (Acta2, Cnn1, and Snai1, T ); EndIT (C1qa, C1qb, Lyz2, and Cd68, U ); and EndFT (Lgals3, Trem2, and Spp1, V ). Shown are mean ± SEM, each dot represents % of confetti + ECs coexpressing FIRE markers in each longitudinal section used for quantification (N=4 to 10 longitudinal sections for RCA; N=6 to 31 longitudinal sections for LCA). P values were calculated by 2-tailed unpaired Student t test with or without Welch’s correction for normal data and 2-tailed unpaired Mann-Whitney U test for non-normal data.
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EC-Confetti mice treated with d-flow and hypercholesterolemia at 4 weeks post-PCL (N=6 male and 15 female) were imaged macroscopically ( A ) and LCAs/RCAs were longitudinally sectioned, stained, imaged by fluorescence microscopy ( B-R ), and quantified ( S-V ). A shows representative gross image of LCA, RCA, and aortic arch. B-R . LCAs and RCAs were immunostained with markers of endothelial inflammation (Vcam1 and Icam1, B-D ); EndMT (Acta2, Snai1, and Cnn1, E-H ); EndIT (Cd68, C1qa, C1qb, and Lyz2, I-M ); and EndFT (Spp1, Lgals3, Trem2, and BODIPY, N-R ). B, E, I, and N show merged images of confetti and FIRE markers at low magnification (10X), while the rest show 40X images. Confetti signals show eGFP (green), YFP (green), and RFP (red). All FIRE markers are shown in white except for green BODIPY ( R ). White arrows indicate confetti + ECs co-expressing the FIRE markers. S-V. Percent confetti + ECs co-expressing each FIRE marker was quantified by a combined <t>Matlab</t> and ImageJ analysis. Confetti + ECs expressing markers of inflammation (Icam1 and Vcam1, S ); EndMT (Acta2, Cnn1, and Snai1, T ); EndIT (C1qa, C1qb, Lyz2, and Cd68, U ); and EndFT (Lgals3, Trem2, and Spp1, V ). Shown are mean ± SEM, each dot represents % of confetti + ECs coexpressing FIRE markers in each longitudinal section used for quantification (N=4 to 10 longitudinal sections for RCA; N=6 to 31 longitudinal sections for LCA). P values were calculated by 2-tailed unpaired Student t test with or without Welch’s correction for normal data and 2-tailed unpaired Mann-Whitney U test for non-normal data.
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EC-Confetti mice treated with d-flow and hypercholesterolemia at 4 weeks post-PCL (N=6 male and 15 female) were imaged macroscopically ( A ) and LCAs/RCAs were longitudinally sectioned, stained, imaged by fluorescence microscopy ( B-R ), and quantified ( S-V ). A shows representative gross image of LCA, RCA, and aortic arch. B-R . LCAs and RCAs were immunostained with markers of endothelial inflammation (Vcam1 and Icam1, B-D ); EndMT (Acta2, Snai1, and Cnn1, E-H ); EndIT (Cd68, C1qa, C1qb, and Lyz2, I-M ); and EndFT (Spp1, Lgals3, Trem2, and BODIPY, N-R ). B, E, I, and N show merged images of confetti and FIRE markers at low magnification (10X), while the rest show 40X images. Confetti signals show eGFP (green), YFP (green), and RFP (red). All FIRE markers are shown in white except for green BODIPY ( R ). White arrows indicate confetti + ECs co-expressing the FIRE markers. S-V. Percent confetti + ECs co-expressing each FIRE marker was quantified by a combined <t>Matlab</t> and ImageJ analysis. Confetti + ECs expressing markers of inflammation (Icam1 and Vcam1, S ); EndMT (Acta2, Cnn1, and Snai1, T ); EndIT (C1qa, C1qb, Lyz2, and Cd68, U ); and EndFT (Lgals3, Trem2, and Spp1, V ). Shown are mean ± SEM, each dot represents % of confetti + ECs coexpressing FIRE markers in each longitudinal section used for quantification (N=4 to 10 longitudinal sections for RCA; N=6 to 31 longitudinal sections for LCA). P values were calculated by 2-tailed unpaired Student t test with or without Welch’s correction for normal data and 2-tailed unpaired Mann-Whitney U test for non-normal data.
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EC-Confetti mice treated with d-flow and hypercholesterolemia at 4 weeks post-PCL (N=6 male and 15 female) were imaged macroscopically ( A ) and LCAs/RCAs were longitudinally sectioned, stained, imaged by fluorescence microscopy ( B-R ), and quantified ( S-V ). A shows representative gross image of LCA, RCA, and aortic arch. B-R . LCAs and RCAs were immunostained with markers of endothelial inflammation (Vcam1 and Icam1, B-D ); EndMT (Acta2, Snai1, and Cnn1, E-H ); EndIT (Cd68, C1qa, C1qb, and Lyz2, I-M ); and EndFT (Spp1, Lgals3, Trem2, and BODIPY, N-R ). B, E, I, and N show merged images of confetti and FIRE markers at low magnification (10X), while the rest show 40X images. Confetti signals show eGFP (green), YFP (green), and RFP (red). All FIRE markers are shown in white except for green BODIPY ( R ). White arrows indicate confetti + ECs co-expressing the FIRE markers. S-V. Percent confetti + ECs co-expressing each FIRE marker was quantified by a combined Matlab and ImageJ analysis. Confetti + ECs expressing markers of inflammation (Icam1 and Vcam1, S ); EndMT (Acta2, Cnn1, and Snai1, T ); EndIT (C1qa, C1qb, Lyz2, and Cd68, U ); and EndFT (Lgals3, Trem2, and Spp1, V ). Shown are mean ± SEM, each dot represents % of confetti + ECs coexpressing FIRE markers in each longitudinal section used for quantification (N=4 to 10 longitudinal sections for RCA; N=6 to 31 longitudinal sections for LCA). P values were calculated by 2-tailed unpaired Student t test with or without Welch’s correction for normal data and 2-tailed unpaired Mann-Whitney U test for non-normal data.

Journal: bioRxiv

Article Title: Disturbed Flow Induces Reprogramming of Endothelial Cells to Immune-like and Foam Cells under Hypercholesterolemia during Atherogenesis

doi: 10.1101/2025.03.06.641843

Figure Lengend Snippet: EC-Confetti mice treated with d-flow and hypercholesterolemia at 4 weeks post-PCL (N=6 male and 15 female) were imaged macroscopically ( A ) and LCAs/RCAs were longitudinally sectioned, stained, imaged by fluorescence microscopy ( B-R ), and quantified ( S-V ). A shows representative gross image of LCA, RCA, and aortic arch. B-R . LCAs and RCAs were immunostained with markers of endothelial inflammation (Vcam1 and Icam1, B-D ); EndMT (Acta2, Snai1, and Cnn1, E-H ); EndIT (Cd68, C1qa, C1qb, and Lyz2, I-M ); and EndFT (Spp1, Lgals3, Trem2, and BODIPY, N-R ). B, E, I, and N show merged images of confetti and FIRE markers at low magnification (10X), while the rest show 40X images. Confetti signals show eGFP (green), YFP (green), and RFP (red). All FIRE markers are shown in white except for green BODIPY ( R ). White arrows indicate confetti + ECs co-expressing the FIRE markers. S-V. Percent confetti + ECs co-expressing each FIRE marker was quantified by a combined Matlab and ImageJ analysis. Confetti + ECs expressing markers of inflammation (Icam1 and Vcam1, S ); EndMT (Acta2, Cnn1, and Snai1, T ); EndIT (C1qa, C1qb, Lyz2, and Cd68, U ); and EndFT (Lgals3, Trem2, and Spp1, V ). Shown are mean ± SEM, each dot represents % of confetti + ECs coexpressing FIRE markers in each longitudinal section used for quantification (N=4 to 10 longitudinal sections for RCA; N=6 to 31 longitudinal sections for LCA). P values were calculated by 2-tailed unpaired Student t test with or without Welch’s correction for normal data and 2-tailed unpaired Mann-Whitney U test for non-normal data.

Article Snippet: To determine the proportion of confetti + ECs undergoing FIRE, multi-level image thresholding was performed via combined use of custom-developed MATLAB algorithms and ImageJ .

Techniques: Staining, Fluorescence, Microscopy, Expressing, Marker, MANN-WHITNEY